Abstract: TH-PO544
Glomerular Dissection Allows High-Throughput RNA Sequencing of Single Podocytes
Session Information
- Glomerular Diseases: Omics, Biomarkers, and Tools
October 24, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology
Authors
- Kim, Najeong, Case Western Reserve University, Cleveland, Ohio, United States
- Jadhav, Darshan, Case Western Reserve University, Cleveland, Ohio, United States
- Garcia, Nestor H., Case Western Reserve University, Cleveland, Ohio, United States
- Gonzalez-Vicente, Agustin, Case Western Reserve University, Cleveland, Ohio, United States
Group or Team Name
- Kidney Precision Medicine Project (KPMP).
Background
Podocytes (POD) are essential for maintaining the integrity of the glomerular filtration barrier. Persistent insult to POD leads to proteinuria and chronic kidney disease. Single nuclei (sn) RNA sequencing (RNAseq) is an important tool in kidney research. As POD represent a small percentage of cells in kidneys, we developed a method to maximize POD recovery.
Methods
Rat glomeruli were microdissected and nuclei suspensions obtained by cellular fractionation. Libraries were prepared with 10X Chromium X technology and sequenced on the Illumina NovaSeq X platform.
Results
Western blots (Fig 1) of 77 glomeruli show enrichment for the slit-diaphragm protein nephrin. We also recovered 22.5 ng RNA from 30 glomeruli with a 28S/18S ratio >1.8. We prepared two sn-libraries, using ~180 (Rat1) and ~90 (Rat2) glomeruli, which yielded 6590 and 1022 sn-transcriptomes, respectively. We quality-controlled and clustered cells in Seurat, and used label transfer from a reference human kidney atlas (PMID: 37468583) to assign cell types (Fig 2). The number of glomerular cells recovered was: POD 1534 (22.2%), parietal epithelial cells (PEC) 96 (1.6%), mesangial cells (MC) 61 (0.9%) and glomerular capillary endothelial cells (EC-GC) 36 (0.5%). The reference atlas contains 2.1% POD, 1.1% PEC, 0.4% MC and 1.3% EC-GC. In addition, the reference atlas recovered on average 73 POD/biopsy, while our pilot study recovered 759 POD/sample.
Conclusion
Collectively, these findings show that our protocol enriches POD proteins and yields high-quality RNA suitable for sequencing. Isolated glomeruli also resulted in a more than tenfold increase in sn-POD transcriptomes per sample compared to kidney biopsies. Further development of this methodology will support high-throughput sequencing of POD with small sample sizes.
Funding
- NIDDK Support